The main goal of our research is to elucidate the neuronal mechanisms responsible for generating locomotor rhythm in mammals. Due to the complexity of the CNS, the communications among individual neurons within such pattern generating networks remain undefined. The proposed work will probe the fundamentals of motor rhythms by explanting a portion of the neonatal mouse spinal cord (with or without the hindlimbs) and recording motor activity while the tissue is maintained in an isolated chamber. Spontaneous activity will be studied as well as activity evoked by electrical and pharmacological stimulation. In addition to short-term in vitro studies, thin (200 - 300 mu (m) transverse sections of cervical and lumbar spinal cord will be maintained in tissue culture for long-term physiological and morphological analyses of spinal networks. As we establish a preparation with a relevant motor rhythm that can provide sufficient access to test individual network components. It becomes increasingly important to know how our in vitro system compares morphologically with the in situ circuitry. Morphometrical characterizations of ventral horn cholinergic cells (e.g., total cholinergic cell counts, cell density measurements and somal size distributions) will be compared between tissue explant cultures and in situ tissue taken from the same litter. After recording intracellularly from ventral horn neurons to obtain information regarding synaptic input during rhythmogenesis, cells will be labeled via intracellular HRP injection and subsequently mapped in relation to their surrounding circuitry using computerized 3-D image analysis. Quantitative autoradiography will be used to map selected amino acid receptors beginning with NMDA receptors. In summary, the proposed studies will help establish a database of morphometric features that can be correlated with the activity patterns produced under a variety of electrical and pharmacological manipulations. It is expected that the numerous experimental approaches involved in characterizing the smallest possible mammalian spinal explants still capable of generating locomotor- like patterns will offer much interest to and many research opportunities for MBRS students.